Studies on patients with methylmalonic aciduria, an inborn error of B12 metabolism, had revealed that in addition to the mitochondrial adenosylcobalamin (AdoCbl)-dependent enzyme, methylmalonyl-CoA mutase, two other disease causing loci were involved. With the recent discovery of the genes encoding MMAA, a G- protein chaperone of unknown function, and adenosyltransferase, which synthesizes AdoCbl from cob(ll)alamin, the mitochondrial components dedicated to assimilation and utilization of B12 have been identified. In contrast, our quest for understanding the interactions and functional interplay between these proteins has just begun and is the subject of this proposal. Methylmalonyl-CoA mutase catalyzes the chemically daunting carbon skeleton rearrangement of methylmalonyl-CoA to succinyl CoA and deploys AdoCbl as a radical reservoir for this reaction. Our studies on this enzyme have furnished insights into how the mutase effects a trillion-fold rate enhancement of Co-C bond homolysis and the role of active site residues in controlling radical reactivity. In this proposal, we plan to address the following specific questions: (i) What is the reaction mechanism of the mutase and how does the bacterial ortholog of MMAA, MeaB, modulate it? We propose to use spectroscopic approaches (EPR, MCD and rapid reaction kinetics) to obtain further mechanistic insights into the mutase reaction and to characterize the influence of MeaB on the mutase reaction coordinate. We also plan to characterize a fusion protein, McmC, in which the mutase and its chaperone, MeaB, are encoded in a single polypeptide and to identify the surfaces of the two proteins that interact with each other, (ii) What are the kinetics of direct transfer of AdoCbl from adenosyltransferase to methylmalonyl-CoA mutase? We will determine and compare the kinetic and thermodynamic parameters associated with B12 binding to the mutase from solution versus its direct transfer from adenosyltransferase to test our model that delivery of B12 is chaperoned, (iii) How do MeaB and MMAA modulate AdoCbl synthesis by adenosyltransferase and its transfer to methylmalonyl-CoA mutase? We will use a combination of ex vivo (with patient cell lines) and in vitro (with a limited number of patient mutations in the mutase and in MMAA) studies to assess the function of the G-protein chaperone and will examine how MeaB modulates the kinetics of direct AdoCbl transfer between adenosyltransferase and the mutase.